Hemostatic compositions

ABSTRACT

The invention discloses a hemostatic composition comprising:
     a) a biocompatible polymer in particulate form suitable for use in hemostasis, and   b) one hydrophilic polymeric component comprising reactive groups.

This application claims the benefit of U.S. Ser. No. 61/545,909 filedOct. 11, 2011, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to hemostatic compositions and processesfor making such compositions.

BACKGROUND OF THE INVENTION

Hemostatic compositions in dry storage-stable form that comprisebiocompatible, biodegradable, dry stable granular material are knowne.g. from WO98/008550A or WO 2003/007845A. These products have beensuccessfully applied on the art for hemostasis. Floseal® is an examplefor a powerful and versatile hemostatic agent consisting of a granulargelatin matrix swollen in a thrombin-containing solution to form aflowable paste.

Since such products have to be applied to humans, it is necessary toprovide highest safety standards for quality, storage-stability andsterility of the final products and the components thereof. On the otherhand, manufacturing and handling should be made as convenient andefficient as possible.

On the other hand, it has been found that previous hemostaticcompositions for wound healing failed to induce hemostasis at conditionswith impaired hemostasis (e.g. after heparinization). It is thereforedesired to provide materials and compositions with improved hemostasis.Moreover, a strong adherence of the compositions applied to the tissueis needed when the composition is applied to a wound. It is also desiredto provide material with suitable swelling behavior after application toa wound.

It is an object of the present invention to overcome such problems andprovide suitable hemostatic compositions with improved adheringproperties and methods for making such hemostatic composition. Thecompositions should also be provided in a convenient and usable manner.The products should preferably be provided in product formats enabling aconvenient provision of “ready-to-use” hemostatic compositions, whichcan be directly applied to an injury without any time consumingreconstitution steps.

SUMMARY OF THE INVENTION

Therefore, the present invention provides a hemostatic compositioncomprising:

-   a) a biocompatible polymer in particulate form suitable for use in    hemostasis, and-   b) one hydrophilic polymeric component comprising reactive groups.

The combination of a biocompatible polymer in particulate form with onehydrophilic polymeric component provides a composition with improvedhemostatic properties and with improved tissue adherence. This isspecifically suitable for wound treatment wherein induction ofhemostasis failed, e.g. at conditions with impaired hemostasis (e.g.after heparinization). The compositions according to the presentinvention improve hemostasis. Furthermore, the compositions according tothe present invention show a strong adherence to the tissue when appliedto a wound.

Upon contact with bleeding tissue, a crosslinking reaction of thehydrophilic polymeric component with the blood proteins leads toformation of a gel with sealing and hemostatic properties. Crosslinkingalso occurs to the tissue surface proteins and, depending on the natureof the biocompatible polymer material, may also occur to thebiocompatible polymer material. The latter reaction contributes to animproved adhesion of the composition material to the wounded tissuesurface.

A further aspect relates to a method of treating an injury comprisingadministering a hemostatic composition to the site of injury.

Also provided is a kit for the treatment of an injury, comprising ahemostatic composition as herein disclosed and instructions for use.

The present invention also refers to a method for producing thehemostatic composition according to the invention in a convenient mannerallowing the composition to be easily at hand for medical use. Theinvention further relates to a method for delivering a hemostaticcomposition to a target site in a patient's body, said method comprisingdelivering a hemostatic composition produced by the process of thepresent invention to the target site. According to another aspect, thepresent invention relates to a finished final container obtained by theprocess according of the present invention containing the presenthemostatic composition. The invention also relates to a method forproviding a ready-to-use hemostatic composition comprising contacting ahemostatic composition produced by the process of the present inventionwith a pharmaceutically acceptable diluent as well as to a kitcomprising the finished final container and other means for applying thecomposition (e.g. a diluent container). The compositions according tothe present invention are particularly useful for providing hemostasisat bleeding sites, including surgical bleeding sites, traumatic bleedingsites and the like. An exemplary use of the compositions may be insealing the tissue tract above a blood vessel penetration created forvascular catheterization.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention provides an improvement in hemostaticcompositions. The hemostatic compositions according to the inventioncontain biocompatible polymers in particulate form, e.g. granules of abiocompatible polymer (e.g. gelatin, fibrin, chitosan, fibronectin,collagen, especially gelatin) suitable for use in hemostasis (the“hemostatic biocompatible polymer component” or the “hemostatic polymer.Admixed to this biocompatible polymer for hemostasis is one hydrophilicpolymeric component comprising reactive groups. According to the presentinvention, the reactive groups of the polymeric component have retainedtheir reactivity until the composition is brought to the place ofclinical action, e.g. on to the wound.

The biocompatible polymers in particulate form suitable for use inhemostasis may include dimensionally isotropic or non-isotropic forms.For example, the biocompatible polymers according to the presentinvention may be granules or fibers; and may be present in discontinuousstructures, for example in powder forms.

According to a preferred embodiment, the biocompatible polymer is liquidabsorbing. For example, upon contact with liquids, e.g. aqueoussolutions or suspensions (especially a buffer or blood) the polymertakes up the liquid and will display a degree of swelling, depending onthe extent of hydration. The material preferably absorbs from about 200%to about 2000%, especially from about 400% to about 1300% wafer oraqueous buffer by weight, corresponding to a nominal increase indiameter or width of an individual particle of subunit in the range frome.g. approximately 50% to approximately 500%, usually from approximately50% to approximately 250%. For example, if the (dry) granular particleshave a preferred size range of 0.01 mm to 1.5 mm, especially of 0.05 mmto 1 mm, the fully hydrated composition (e.g. after administration on awound or after contact with an aqueous buffer solution) may have a sizerange of 0.05 mm to 3 mm, especially of 0.25 mm to 1.5 mm.

The equilibrium swell of preferred biocompatible polymers of the presentinvention may generally range e.g. from 400% to 1300%, preferably beingfrom 500% to 1100%, depending on its intended use. Such equilibriumswell may be controlled e.g. (for a crosslinked polymer) by varying thedegree of crosslinking, which in turn is achieved by varying thecrosslinking conditions, such as the type of crosslinking method,duration of exposure of a crosslinking agent, concentration of acrosslinking agent, crosslinking temperature, and the like. Materialshaving differing equilibrium swell values perform differently indifferent applications. For example, the ability to inhibit bleeding ina liver divot model was most readily achieved with crosslinked gelatinmaterials having a swell in the range from 700% to 950%. For a femoralartery plug, lower equilibrium swell values in the range from 500% to600% were more successful. Thus, the ability to control crosslinking andequilibrium swell allows the compositions of the present invention to beoptimized for a variety of uses. In addition to equilibrium swell, it isalso important to control the hydration of the material immediatelyprior to delivery to a target site. Hydration and equilibrium swell are,of course, intimately connected. A material with 0% hydration will benon-swollen. A material with 100% hydration will be at its equilibriumwater content. Hydrations between 0% and 100% will correspond toswelling between the minimum and maximum amounts.

According to a preferred embodiment of the present invention, thebiocompatible polymer and the hydrophilic polymeric component arepresent in dry form, preferably in mixed dry form.

The biocompatible polymer in particulate form suitable for use inhemostasis of the present invention may be formed from biologic andnon-biologic polymers. Suitable biologic polymers may contain a protein,a polysaccharide, a biologic polymer, a non-biologic polymer; andderivatives and combinations thereof. Suitable proteins include gelatin,collagen, albumin, hemoglobin, fibrinogen, fibrin, casein, fibronectin,elastin, keratin, and laminin; and derivatives and combinations thereof.Particularly preferred is the use of gelatin or soluble non-fibrillarcollagen, more preferably gelatin, and exemplary gelatin formulationsare set forth below. Other suitable biologic polymers includepolysaccharides, such as glycosaminoglycans, starch, cellulose, dextran,hemicellulose, xylan, agarose, alginate and chitosan; and derivativesand combinations thereof. Suitable non-biologic polymers will beselected to be degradable by either of two mechanisms, i.e. (1) breakdown of the polymeric backbone or (2) degradation of side chains whichresult in aqueous solubility. Exemplary non-biologic biocompatiblepolymers suitable for use in hemostasis include synthetics, such aspolyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides,polyethyleneimines, polyvinyl resins, polylactide-glycolides,polycaprolactones, and polyoxyethlenes; and derivatives and combinationsthereof. Also combinations of different kinds of polymers are possible(e.g. proteins with polysaccharides, proteins with non-biologichydrogel-forming polymers, etc.). Preferred hemostatic polymers compriseamino-groups, specifically if the hydrophilic polymeric component hasreactive groups which react with amino-groups upon administration (e.g.in the wound environment).

“A derivative thereof” includes any chemically modified polymer, such ase.g. a crosslinked polymer.

Preferred hemostatic polymers comprise nucleophilic groups, such as e.g.amino-groups, specifically if the hydrophilic polymeric component hasreactive groups which react with amino-groups upon administration (e.g.in the wound environment).

According to a preferred embodiment of the present invention, thebiocompatible polymer is selected from the group consisting of gelatin,collagen, albumin, fibrinogen, fibrin and derivatives thereof (asdefined above); especially preferred the polymer is gelatin or collagen;especially preferred is crosslinked gelatin.

According to a preferred embodiment of the present invention, thebiocompatible polymer suitable for use in hemostasis contains acrosslinked protein, a crosslinked polysaccharide, a crosslinkedbiologic polymer, a crosslinked non-biologic polymer; or mixturesthereof.

A non-crosslinked polymer may be crosslinked in any manner suitable toreconstitute, e.g. to form a suitable hydrogel base of the hemostaticpolymer. For example, polymeric molecules may be crosslinked using bi-or poly-functional crosslinking agents which covalently attach to two ormore polymer molecules chains. Exemplary bifunctional crosslinkingagents include aldehydes, epoxides, succinimides, carbodiimides,maleimides, azides, carbonates, isocyanates, divinyl sulfone, alcohols,amines, imidates, anhydrides, halides, silanes, diazoacetate,aziridines, and the like. Alternatively, crosslinking may be achieved byusing oxidizers and other agents, such as periodates, which activateside-chains or moieties on the polymer so that they may react with otherside-chains or moieties to form the crosslinking bonds. An additionalmethod of crosslinking comprises exposing the polymers to radiation,such as gamma radiation, to activate the polymer chains to permitcrosslinking reactions. Dehydrothermal crosslinking methods may also besuitable. Preferred methods for crosslinking gelatin molecules aredescribed below.

The biocompatible hemostatic polymer—once applied to a wound—forms anefficient matrix which can form a barrier for blood flow. Specificallythe swelling properties of the hemostatic polymer can make it aneffective mechanical barrier against bleeding and rebleeding processes.

In a preferred embodiment, the hemostatic compositions according to thepresent invention are provided or used as granular preparations.According to a preferred embodiment, the biocompatible polymergranulates suitable for use in hemostasis contain a crosslinked protein,a crosslinked polysaccharide, or a crosslinked non-biologic polymer; ormixtures thereof.

As mentioned above, the biocompatible polymer suitable for use inhemostasis is preferably a granular material. This granular material canrapidly swell when exposed to a fluid (i.e. the diluent) and in thisswollen form is capable of contributing to a flowable paste that can beapplied to a bleeding site. The biocompatible polymer, e.g. gelatin, maybe provided as a film which can then be milled to form a granularmaterial. Most of the particles contained in this granular material(e.g. more than 90% w/w) have preferably particle sizes of 10 to 1,000μm, especially 50 to 700 μm.

According to a preferred embodiment, the biocompatible polymer inparticulate form suitable for use in hemostasis is a crosslinkedgelatin. Dry crosslinked gelatin powder can be prepared to re-hydraterapidly if contacted with a pharmaceutically acceptable diluent. Thegelatin granules, especially in the form of a gelatin powder, preferablycomprise relatively large particles, also referred to as fragments orsub-units, as described in WO98/08550A and WO2003/007845A. A preferred(median) particle size will be the range from 10 to 1,000 μm, preferablyfrom 50 to 700 μm, but particle sizes outside of this preferred rangemay find use in many circumstances. The dry compositions will alsodisplay a significant “equilibrium swell” when exposed to an aqueousre-hydrating medium (=diluents, also referred to as reconstitutionmedium or re-hydration medium). Preferably, the swell will be in therange from 400% to 1000%. “Equilibrium swell” may be determined bysubtracting the dry weight of the gelatin hydrogel powder from itsweight when fully hydrated and thus fully swelled. The difference isthen divided by the dry weight and multiplied by 100 to give the measureof swelling. The dry weight should be measured after exposure of thematerial to an elevated temperature for a time sufficient to removesubstantially all residual moisture, e.g., two hours at 120° C. Theequilibrium hydration of the material can be achieved by immersing thedry material in a pharmaceutically acceptable diluent, such as aqueoussaline, for a time period sufficient for the water content to becomeconstant, typically for from 18 to 24 hours at room temperature.

Exemplary methods for producing crosslinked gelatins are as follows.Gelatin is obtained and suspended in an aqueous solution to form anon-crosslinked hydrogel, typically having a solids content from 1% to70% by weight, usually from 3% to 10% by weight. The gelatin iscrosslinked, typically by exposure to either glutaraldehyde (e.g., 0.01%to 0.05% w/w, overnight at 0° C. to 15° C. in aqueous buffer), sodiumperiodate (e.g., 0.05 M, held at 0° C. to 15° C. for 48 hours) or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (“EDC”) (e.g., 0.5% to1.5% w/w overnight at room temperature), or by exposure to about 0.3 to3 megarads of gamma or electron beam radiation. Alternatively, gelatinparticles can be suspended in an alcohol, preferably methyl alcohol orethyl alcohol, at a solids content of 1% to 70% by weight, usually 3% to10% by weight, and crosslinked by exposure to a crosslinking agent,typically glutaraldehyde (e.g., 0.01% to 0.1% w/w, overnight at roomtemperature). In the case of aldehydes, the pH should be held from about6 to 11, preferably from 7 to 10. When crosslinking with glutaraldehyde,the crosslinks are formed via Schiff bases which may be stabilized bysubsequent reduction, e.g., by treatment with sodium borohydride. Aftercrosslinking, the resulting granules may be washed in water andoptionally rinsed in an alcohol, and dried. The resulting dry powdersmay then be provided in the final container as described herein.

Preferably, the biocompatible polymer is provided in a dry granular formfor producing the hemostatic compositions according to the presentinvention. A “dry granular preparation of a biocompatible polymer”according to the present invention is known e.g. from WO 98/08550 A.Preferably, the polymer is a biocompatible, biodegradable dry stablegranular material.

The dry polymer according to the present invention Is usually providedwith particle sizes of 10 to 1,000 μm. Usually, the polymer particleshave a mean particle diameter (“mean particle diameter” is the mediansize as measured by laser diffractometry; “median size” (or mass medianparticle diameter) is the particle diameter that divides the frequencydistribution in half; fifty percent of the particles of a givenpreparation have a larger diameter, and fifty percent of the particleshave a smaller diameter) from 10 to 1000 μm, especially 50 to 700 μm(median size). Applying larger particles is mainly dependent on themedical necessities; particles with smaller mean particle diameters areoften more difficult to handle in the production process. The drypolymer is therefore provided in granular form. Although the termspowder and granular (or granulates) are sometimes used to distinguishseparate classes of material, powders are defined herein as a specialsub-class of granular materials. In particular, powders refer to thosegranular materials that have the finer grain sizes, and that thereforehave a greater tendency to form clumps when flowing. Granules includecoarser granular materials that do not tend to form clumps except whenwet. For the present application the particles used are those which canbe coated by suitable coating techniques Particle size of the polymergranules according to the present invention can therefore easily beadapted and optimized to a certain coating technique by the necessitiesof this technique.

The hydrophilic polymeric component (also referred to as “reactivehydrophilic component” or “hydrophilic (polymeric) crosslinker”) of thehemostatic composition according to the present invention is ahydrophilic crosslinker which is able to react with its reactive groupsonce the hemostatic composition is applied to a patient (e.g. to a woundof a patient or another place where the patient is in need of ahemostatic activity). Therefore it is important for the presentinvention that the reactive groups of the polymeric component arereactive when applied to the patient. It is therefore necessary tomanufacture the hemostatic composition according to the presentinvention so that the reactive groups of the polymeric component whichshould react once they are applied to a wound are retained during themanufacturing process.

This can be done in various ways. For example, usual hydrophilicpolymeric components have reactive groups which are susceptible tohydrolysis after contact with water. Accordingly, premature contact withwater or aqueous liquids has to be prevented before administration ofthe hemostatic composition to the patient, especially duringmanufacture. However, processing of the hydrophilic polymeric componentduring manufacturing may be possible also in an aqueous medium atconditions where the reactions of the reactive groups are inhibited(e.g. at a low pH). If the hydrophilic polymeric components can bemelted, the melted hydrophilic polymeric components can be sprayed orprinted onto the matrix of the biopolymer. It is also possible to mix adry form (e.g. a powder) of the hydrophilic polymeric component with adry form of the biocompatible polymer suitable for use in hemostasis. Ifnecessary, then an increase of the temperature can be applied to meltthe sprinkled hydrophilic polymeric component to the biocompatiblepolymer suitable for use in hemostasis to achieve a permanent coating ofthe hemostatic composition. Alternatively, these hydrophilic polymericcomponents can be taken up into inert organic solvents (inert vis-à-visthe reactive groups of the hydrophilic polymeric components) and broughtonto the matrix of the biomaterial. Examples of such organic solventsare dry ethanol, dry acetone or dry dichloromethane (which are e.g.inert for hydrophilic polymeric components, such as NHS-estersubstituted PEGs).

The term “one hydrophilic polymeric component comprising reactivegroups” means that the presence of a second or further hydrophilicpolymeric component with nucleophilic reactive groups is excluded in ahemostatic composition according to the present invention.

In a preferred embodiment the hydrophilic polymer component is a singlehydrophilic polymer component and is a polyalkylene oxide polymer,preferably a PEG comprising polymer. The reactive groups of thisreactive polymer are preferably electrophilic groups.

The reactive hydrophilic component may be a multi-electrophilicpolyalkylene oxide polymer, e.g, a multi-electrophilic PEG. The reactivehydrophilic component can include two or more electrophilic groups,preferably a PEG comprising two or more reactive groups selected fromsuccinimidylesters (—CON(COCH₂)₂), aldehydes (—CHO) and isocyanates(—N═C═O), e.g, a component as disclosed in the WO2008/016983 A(incorporated herein by reference in its entirety) and one of thecomponents of the commercially available ones under the trademarkCoSeal®.

Preferred electrophilic groups of the hydrophilic polymeric crosslinkeraccording to the present invention are groups reactive to the amino-,carboxy-, thiol- and hydroxy-groups of proteins, or mixtures thereof.

Preferred amino group-specific reactive groups are NHS-ester groups,imidoester groups, aldehyde-groups, carboxy-groups in the presence ofcarbodiimides, isocyanates, or THPP (beta-[Tris(hydroxymethyl)phosphino]propionic acid), especially preferred isPentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate(=Pentaerythritoltetrakis[1-1′-oxo-5′-succinimidylpentanoate-2-poly-oxoethyleneglycole]ether(=an NHS-PEG with MW 10,000).

Preferred carboxy-group specific reactive groups are amino-groups in thepresence of carbodiimides.

Preferred thiol group-specific reactive groups are maleimides orhaloacetyls.

Preferred hydroxy group-specific reactive group is the isocyanate group.The reactive groups on the hydrophilic crosslinker may be identical(homofunctional) or different (heterofunctional). The hydrophilicpolymeric component can have two reactive groups (homobifunctional orheterobifunctional) or more (homo/hetero-trifunctional or more).

In special embodiments the material is a synthetic polymer, preferablycomprising PEG. The polymer can be a derivative of PEG comprising activeside groups suitable for crosslinking and adherence to a tissue.

By the reactive groups the hydrophilic reactive polymer has the abilityto crosslink blood proteins and also tissue surface proteins.Crosslinking to the biomaterial is also possible.

The multi-electrophilic polyalkylene oxide may include two or moresuccinimidyl groups. The multi-electrophilic polyalkylene oxide mayinclude two or more maleimidyl groups.

Preferably, the multi-electrophilic polyalkylene oxide is a polyethyleneglycol or a derivative thereof.

In a most preferred embodiment the hydrophilic polymeric component ispentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate(═COH102, also pentaerythritoltetrakis[1-1′-oxo-5′-succinimidylpentanoate-2-poly-oxoethyleneglycole]ether).

The hydrophilic polymeric component is a hydrophilic crosslinker.According to a preferred embodiment, this crosslinker has more than tworeactive groups for crosslinking (“arms”), for example three, four,five, six, seven, eight, or more arms with reactive groups forcrosslinking. For example, NHS-PEG-NHS is an effective hydrophiliccrosslinker according to the present invention. However, for someembodiments, a 4-arm polymer (e.g. 4-arms-p-NP-PEG) may be morepreferred; based on the same rationale, an 8-arm polymer (e.g.8-arms-NHS-PEG) may even be more preferred for those embodiments wheremulti-reactive crosslinking is beneficial. Moreover, the hydrophiliccrosslinker according to the present invention is a polymer, i.e. alarge molecule (macromolecule) composed of repeating structural unitswhich are typically connected by covalent chemical bonds. Thehydrophilic polymer component according to the present invention shouldhave a molecular weight of at least 1000 Da (to properly serve ascrosslinker in the hemostatic composition according to the presentinvention); preferably the crosslinking polymers according to thepresent invention has a molecular weight of at least 5000 Da, especiallyof at least 8000 Da.

For some hydrophilic crosslinkers, the presence of basic reactionconditions (e.g. at the administration site) is preferred or necessaryfor functional performance (e.g. for a faster crosslinking reaction atthe administration site). For example, carbonate or bicarbonate ions(e.g. as a buffer with a pH of 7.6 or above, preferably of 8.0 or above,especially of 8.3 and above) may be additionally provided at the site ofadministration (e.g. as a buffer solution or as a fabric or pad soakedwith such a buffer), so as to allow an improved performance of thehemostatic composition according to the present invention or to allowefficient use as a hemostatic and/or wound adherent material.

The reactivity of the hydrophilic polymeric component (which, asmentioned, acts as a crosslinker) in the composition according to thepresent invention is retained in the composition. This means that thereactive groups of the crosslinker have not yet reacted with thehemostatic composition and are not hydrolyzed by water (or at least notin a significant amount which has negative consequences on thehemostatic functionality of the present compositions). This can beachieved by combining the hemostatic polymer with the hydrophiliccrosslinker in a way which does not lead to reaction of the reactivegroups of the crosslinker with the hemostatic polymer or with water.Usually, this includes the omitting of aqueous conditions (or wetting),especially wetting without the presence of acidic conditions (ifcrosslinkers are not reactive under acidic conditions). This allows theprovision of reactive hemostatic materials.

According to a specifically preferred hemostatic composition of theinvention, the biocompatible polymer is crosslinked gelatin and thehydrophilic polymeric component ispentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate.

Preferred ratios of the biocompatible polymer to hydrophilic polymericcomponent in the hemostatic composition according to the presentinvention are from 0.1 to 50% w/w, preferably from 5 to 40% w/w.

The hemostatic compositions according to the present invention arepreferably provided as dry composition, e.g. as a physical mixture, ofthe hemostatic polymer and the hydrophilic reactive component, whereinthe biocompatible polymer and the hydrophilic polymeric component arepresent in dry form, preferably in mixed dry form. “Mixed” according tothe present invention includes powder mixing, coating, impregnating,blending, agglomerating, co-lyophilizing, drying from suspension,subsequent or concurrent co-filling, co-extruding, etc.

A “dry” hemostatic composition according to the present invention hasonly a residual content of moisture which may approximately correspondto the moisture content of comparable available products, such asFloseal® (Floseal, for example, has about 12% moisture as a dryproduct). Usually, the dry composition according to the presentinvention has a residual moisture content below these products,preferably below 10% moisture, more preferred below 5% moisture, morepreferred below 2.5%, especially below 1% moisture. The hemostaticcomposition according to the present invention can also have lowermoisture content, e.g. 0.1% or even below. Preferred moisture contentsof the dry hemostatic composition according to the present invention are0.1 to 10%, especially 0.5 to 5%. It is clear that the dryer thecomposition is, the longer their shelf life is and the lower is the riskthat the hemostatic properties of the composition as a whole suffer.

As already stated, the biocompatible polymer in particulate formsuitable for use in hemostasis is preferably gelatin in powder form,especially wherein the powder particles have a median particle size of10 to 1000 μm, preferably from 50 to 750 μm, more preferred from 150 to700 μm, especially from 150 to 500 μm.

Further components may be present in the hemostatic compositionaccording to the present invention. According to preferred embodiments,the hemostatic compositions according to the present invention mayfurther comprise a substance selected from the group consisting ofantifibrinolytic, procoagulant, platelet activator, antibiotic,vasoconstrictor, dye, growth factors, bone morphogenetic proteins andpain killers.

The hemostatic composition according to the present invention maycomprise a further composition of gelatin and a polyvalent nucelophilicsubstance, preferably human serum albumin, optionally at a basic pH(e.g. pH 8 to 11, preferably 9 to 10, especially at a pH of 9.5). The 2components may then be co-applied to an injury.

According to another aspect, the present invention relates to the use ofa hemostatic composition according to the present invention for thetreatment of an injury selected from the group consisting of a wound, ahemorrhage, damaged tissue, bleeding tissue and/or bone defect.

The present invention also relates to a method of treating an injuryselected from the group consisting of a wound, a hemorrhage, damagedtissue and/or bleeding tissue comprising administering a hemostaticcomposition according to the present invention to the site of injury.

According to another aspect, the present invention provides a kit forthe treatment of an injury selected from the group consisting of awound, a hemorrhage, damaged tissue and/or bleeding tissue comprising

-   a) a hemostatic composition according to the present invention; and-   b) instructions for use

The present invention also relates to a method for producing ahemostatic composition according to the present invention comprising thestep of mixing, a biocompatible polymer suitable for use in hemostasisand one hydrophilic polymeric component comprising reactive groups indry form.

It is preferred to provide the hemostatic compositions according to thepresent invention in dry form in an administration container, preferablyin a syringe, optionally together with a pharmaceutically acceptablediluent.

These hemostatic compositions according to the present invention may bereconstituted to “ready-to-use” hemostatic preparations usingpharmaceutically acceptable diluents (e.g. aqueous ionic solutions).Preferably, the “ready-to use” preparations are present or provided ashydrogels. Products of this kind are known in principle in the art, yetin a different format. Usually, the components are provided as separateentities in dry form. Before mixing the components for administration toa patient, the dry components are usually contacted separately withpharmaceutically acceptable diluents. Mixing of the components is thenperformed by mixing the separately reconstituted components.

For stability reasons, such products (as well as the products accordingto the present invention) are usually provided in a dry form and broughtinto the “ready-to-use” form (which is usually in the form of a(hydro-)gel, suspension or solution) immediately before use,necessitating the addition of wetting or solvation (suspension) agents.

According to the present invention, the hemostatic composition isprovided in dry form in the final container. In the dry form,degradation or inactivation processes for the components aresignificantly and appropriately reduced to enable storage stability.

The dry hemostatic compositions according to the present invention areusually reconstituted (re-hydrated) before use by contacting the drycomposition with a pharmaceutically acceptable diluent. Such apharmaceutically acceptable diluent may be part of the kit according tothe present invention (together with the hemostatic composition). Thediluent according to the present invention may be any suitablereconstitution medium (“reconstitution solution” or “re-hydrationmedium”) for the dry hemostatic composition which allows suitablewetting of the dry composition. Preferably, the dry hemostaticcomposition is reconstituted into a hydrogel as a “ready-to-use” format.

Suitable diluents are pharmaceutically acceptable aqueous fluids, e.g.pharmaceutical grade de-ionized water (if all ionic or buffer componentsare already provided in the dry composition; “water-for-injection”) orpharmaceutical grade aqueous solutions containing specific ions and/orbuffers. Preferably, the diluent comprises a substance selected from thegroup consisting of NaCl, CaCl₂ and sodium acetate (or, of course,mixtures thereof).

For example, a suitable diluent comprises water for injection,and—independently of each other—50 to 200 mM NaCl (preferably 150 mM),10 to 80 mM CaCl₂ (preferably 40 mM) and 1 to 50 mM sodium acetate(preferably 20 mM). Preferably, the diluent can also include a buffer orbuffer system so as to buffer the pH of the reconstituted drycomposition, preferably at a pH of 3.0 to 10.0, more preferred of 6.4 to7.5, especially at a pH of 8.9 to 7.1.

According to a preferred embodiment, the diluent further comprisesthrombin, preferably 10 to 1000 I.U. thrombin/ml, especially 250 to 700I.U. thrombin/ml. Preferably, the hemostatic composition in this readyto use form contains 10 to 100,000 International Units (I.U.) ofthrombin, more preferred 100 to 10,000 I.U., especially 500 to 5,000I.U. The thrombin concentration in the ready-to-use composition ispreferably in the range of 10 to 10,000 I.U., more preferred of 50 to5,000 I.U., especially of 100 to 1,000 I.U./ml. The diluent is used inan amount to achieve the desired end-concentration in the ready-to-usecomposition. The thrombin preparation may contain other usefulcomponent, such as ions, buffers, excipients, stabilizers, etc.

These aqueous diluents may further contain other ingredients, such asexcipients. An “excipient” is an inert substance which is added to thesolution, e.g. to ensure that thrombin retains its chemical stabilityand biological activity upon storage (or sterilization (e.g. byirradiation)), or for aesthetic reasons e.g. color. Preferred excipientsinclude human albumin and sodium acetate. Preferred concentrations ofhuman albumin in the reconstituted product are from 0.1 to 100 mg/ml,preferably from 1 to 10 mg/m. Preferred sodium acetate concentrationsare in the range of from 1 to 10 mg/ml, especially 2 to 5 mg/ml.

Preferably, the thrombin preparation contains human albumin. Preferredsalts are NaCl and/or CaCl₂, both used in the usual amounts andconcentrations applied for thrombin (e.g. 0.5 to 1.5% NaCl (e.g. 0.9%)and/or 20 to 80 mM CaCl₂ (e.g. 40 mM)).

In a preferred embodiment, the pharmaceutically acceptable diluent isprovided In a separate container. This can preferably be a syringe. Thediluent in the syringe can then easily be applied to the final containerfor reconstitution of the dry hemostatic compositions according to thepresent invention. If the final container is also a syringe, bothsyringes can be finished together in a pack. It is therefore preferredto provide the dry hemostatic compositions according to the presentinvention in a syringe which is finished with a diluent syringe with apharmaceutically acceptable diluent for reconstituting said dry andstable hemostatic composition.

According to a preferred embodiment, the final container furthercontains an amount of a stabilizer effective to inhibit modification ofthe polymer when exposed to the sterilizing radiation, preferablyascorbic acid, sodium ascorbate, other salts of ascorbic acid, or anantioxidant.

According to another aspect, the present invention also provides amethod for delivering a hemostatic composition according to theinvention to a target site in a patient's body, said method comprisingdelivering a hemostatic composition produced by the process according tothe present invention to the target site. Although in certainembodiments, also the dry composition can be directly applied to thetarget site (and, optionally be contacted with the diluent a the targetsite, if necessary), it is preferred to contact the dry hemostaticcomposition with a pharmaceutically acceptable diluent beforeadministration to the target site, so as to obtain a hemostaticcomposition in a wetted form, especially a hydrogel form.

The present invention also refers to a finished final container obtainedby the process according to the present invention. This finishedcontainer contains the combined components in a sterile, storage-stableand marketable form. The final container can be any container suitablefor housing (and storing) pharmaceutically administrable compounds.Syringes, vials, tubes, etc. can be used; however, providing thehemostatic compositions according to the present invention in a syringeis specifically preferred. Syringes have been a preferred administrationmeans for hemostatic compositions as disclosed in the prior art alsobecause of the handling advantages of syringes in medical practice. Thecompositions may then preferably be applied (after reconstitution) viaspecific needles of the syringe or via suitable catheters. Thereconstituted hemostatic compositions (which are preferablyreconstituted to form a hydrogel) may also be applied by various othermeans e.g. by a spatula, a brush, a spray, manually by pressure, or byany other conventional technique. Administration of the reconstitutedhemostatic composition to a patient by spraying is specificallypreferred. Usually, the reconstituted hemostatic compositions accordingto the present invention will be applied using a syringe or similarapplicator capable of extruding the reconstituted composition through anorifice, aperture, needle, tube, or other passage to form a bead, layer,or similar portion of material. Mechanical disruption of thecompositions can be performed by extrusion through an orifice in thesyringe or other applicator, typically having a size in the range from0.01 mm to 5.0 mm, preferably 0.5 mm to 2.5 mm. Preferably, however, thehemostatic composition will be initially prepared from a dry form havinga desired particle size (which upon reconstitution, especially byhydration, yields subunits of the requisite size (e.g. hydrogelsubunits)) or will be partially or entirely mechanically disrupted tothe requisite size prior to a final extrusion or other application step.It is, of course evident, that these mechanical components have to beprovided in sterile form (inside and outside) in order to fulfill safetyrequirements for human use.

Another aspect of the invention concerns a method for providing aready-to-use hemostatic composition comprising contacting a hemostaticcomposition produced by the process according to the present inventionwith a pharmaceutically acceptable diluent.

The present invention also concerns a kit comprising the dry and stablehemostatic composition according to the present invention in finishedform and a container with a suitable diluent. Further components of thekit may be instructions for use, administration means, such as syringes,catheters, brushes, etc. (if the compositions are not already providedin the administration means) or other components necessary for use inmedical (surgical) practice, such as substitute needles or catheters,extra vials or further wound cover means. Preferably, the kit accordingto the present invention comprises a syringe housing the dry and stablehemostatic composition and a syringe containing the diluent (or providedto take up the diluent from another diluent container). Preferably,these two syringes are provided in a form adapted to each other so thatthe diluent can be delivered to the dry hemostatic composition byanother entry than the outlet for administering the reconstitutedcomposition.

Therefore, a method for providing a ready to use form of a hemostaticcomposition according to the present invention, wherein the hemostaticcomposition is provided in a first syringe and a diluent forreconstitution is provided in a second syringe, the first and the secondsyringe are connected to each other, and the diluent is brought into thefirst syringe to produce a flowable form of the hemostatic composition;and optionally returning the flowable form of the hemostatic compositionto the second syringe at least once, is a preferred embodiment of thepresent invention. This process (also referred to as “swooshing”)provides a suitable “ready-to-use” form of the compositions according tothe present invention which can easily and efficiently be made alsowithin short times, eg. in emergency situations during surgery. Thisflowable form of the hemostatic composition provided by such a method isspecifically suitable for use in the treatment of an injury selectedfrom the group consisting of a wound, a hemorrhage, damaged tissue,bleeding tissue and/or bone defects.

The invention is further described in the examples below and the drawingfigures, yet without being restricted thereto.

FIG. 1 shows crosslinked gelatin mixed with 20 wt % of NHS-PEG hydratedwith saline solution at neutral pH (Example 1) in a liver punch lesionmodel 5 min post application.

EXAMPLES Example 1 Mixture Neutral

A mixture was prepared by mixing a specific amount of crosslinkedgelatin particles with 20 wt % of NHS-PEG. Typically, 6g of gelatinparticles in a 50 ml test tube were mixed with 1.2 g of NHS-PEG usingend-over-end-mixer for at least 30 minutes in order to obtain ahomogenous mixture of both components. From the mixture obtained, 0.96 gwere weighted in a 5 ml syringe. As a diluent 3.5 ml of saline solutionin a 5 ml syringe with female luer connector were used to hydrate thepowder component before application to a bleeding site.

Hydration of the particulate component with the diluent was achieved byconnection of both syringes and transforming the diluent to the syringefilled with the gelatin. In order to obtain a homogenous product, thecontent of the syringes was pushed back and forth at least 21 times.After hydration, a product obtained was allowed to hydrate for 2minutes. A product obtained was applied to a bleeding wound usingappropriate applicator tip attached to the syringe with a male luer.

Example 2 Mixture Basic

In order to obtain a faster reactive flowable hemostat the mixture asdescribed in Example 1 was hydrated by using 3.5 ml of a basic bufferhaving pH of 9.5 as a diluent.

A product obtained was allowed to hydrate for 2 minutes and was appliedto a blending wound.

Example 3 Mixture Acidic

In order to obtain a reactive flowable hemostat with prolonged stabilitythe mixture as described in Example 1 was hydrated with 3.5 ml of salinesolution having pH adjusted to 1.5 with 1 M of HCl as a diluent.

A product obtained was allowed to hydrate for 2 minutes and was appliedto a bleeding wound.

Example 4 In Vivo Study

A preparation of Example 1 was tested for hemostatic efficacy onheparinized animal (pig) in a punch or biopsy liver lesion. Each lesionin the series was topically treated with the product applied from thesyringe through applicator tip. Moistened gauze was used to helpapproximate the test product to the lesion and the timer was started. Asaline moistened approximation gauze was removed after 30 seconds andthe degree of bleeding was assessed at 30 seconds, 1, 2, 5 and 10minutes after the test articles were applied. Product saturated withblood but without active bleeding was scored as 0. Saline solution wasused to irrigate the excess test articles away from the lesions afterthe 5 minutes assessment. Performance of selected formulations at 5minutes assessment is shown in FIG. 1.

All patent filings, scientific journals, books, treatises, and otherpublications and materials discussed in this application are herebyincorporated by reference for all purposes. While exemplary embodimentshave been described in some detail, by way of example and for clarity ofunderstanding, those of skill in the art will recognize that a varietyof modification, adaptations, and changes may be employed. Hence, thescope of the present invention should be limited solely by the claims.

1. A hemostatic composition comprising: a) a biocompatible polymer inparticulate form suitable for use in hemostasis; and b) one hydrophilicpolymeric component comprising reactive groups.
 2. The hemostaticcomposition according to claim 1, wherein the biocompatible polymer andthe hydrophilic polymeric component are present in dry form.
 3. Thehemostatic composition according to claim 1, wherein said biocompatiblepolymer suitable for use in hemostasis contains a member selected fromthe group consisting of a protein, a polysaccharide, a biologic polymer,a non-biologic polymer; and derivatives and combinations thereof.
 4. Thehemostatic composition according to claim 1, wherein said biocompatiblepolymer suitable for use in hemostasis contains a protein selected fromthe group consisting of gelatin, collagen, albumin, hemoglobin,fibrinogen, fibrin, casein, fibronectin, elastin, keratin, and laminin;and derivatives and combinations thereof.
 5. The hemostatic compositionaccording to claim 1, wherein said biocompatible polymer suitable foruse in hemostasis contains a crosslinked protein, a crosslinkedpolysaccharide, a crosslinked biologic polymer, a crosslinkednon-biologic polymer; or mixtures thereof.
 6. The hemostatic compositionaccording to claim 1, wherein the hydrophilic polymer component is apolyalkylene oxide polymer.
 7. The hemostatic composition according toclaim 1, wherein said hydrophilic polymeric component with reactivegroups is a polyethylene glycol (PEG).
 8. The hemostatic compositionaccording to claim 1, wherein the biocompatible polymer is crosslinkedgelatin and the hydrophilic polymeric component ispentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate. 9.The hemostatic composition according to claim 1, provided in dry form inan administration container.
 10. A method of treating a patient,comprising administering to the patient a hemostatic compositionaccording to claim 1 for the treatment of an injury selected from thegroup consisting of a wound, a hemorrhage, a damaged tissue, a bleedingtissue, and a bone defect.
 11. A method of treating an injury selectedfrom the group consisting of a wound, a hemorrhage, a damaged tissue,and a bleeding tissue comprising administering the hemostaticcomposition of claim 1 to the injury.
 12. A method for producing ahemostatic composition according to claim 1 comprising the step ofmixing a biocompatible polymer suitable for use in hemostasis and onehydrophilic polymeric component comprising reactive groups in dry form.13. A kit comprising a hemostatic composition in dry form according toclaim 1 and a diluent for reconstitution of the hemostatic composition.14. A method for providing a ready to use form of a hemostaticcomposition according to claim 1, wherein the hemostatic composition isprovided in a first syringe and a diluent for reconstitution is providedin a second syringe, the first and the second syringe are connected toeach other, and the diluent is brought into the first syringe to producea flowable form of the hemostatic composition.
 15. The hemostaticcomposition according to claim 2, wherein the biocompatible polymer andthe hydrophilic polymeric component are present in mixed dry form. 16.The hemostatic composition according to claim 6, wherein thepolyalkylene oxide polymer is a PEG comprising polymer.
 17. Thehemostatic composition according to claim 6, wherein the polyalkyleneoxide polymer is a multi-electrophilic polyalkylene oxide polymer. 18.The hemostatic composition according to claim 6, wherein thepolyalkylene oxide polymer is a multi-electrophilic PEG.
 19. Thehemostatic composition according to claim 6, wherein the polyalkyleneoxide polymer is pentaerythritolpoly(ethyleneglycol)ethertetrasuccinimidyl glutarate.
 20. The hemostatic composition according toclaim 7, wherein the hydrophilic polymeric component with reactivegroups is a polyethylene glycol (PEG) comprising two or more reactivegroups selected from succinimidylesters (—CON(COCH₂)₂), aldehydes (—CHO)and isocyanates (—N═C═O).